WO2020254303A1

WO2020254303A1 - Methods and apparatuses for checking the confocality of a scanning and descanning microscope assembly

Info

Publication number: WO2020254303A1
Application number: PCT/EP2020/066589
Authority: WO
Inventors: Joachim Fischer; Matthias Henrich
Original assignee: Abberior Instruments Gmbh
Priority date: 2019-06-19
Filing date: 2020-06-16
Publication date: 2020-12-24
Also published as: US20220113524A1; CN113994246B; JP2022529078A; US11493744B2; DE102019116626B4; JP7168798B2; EP3987335C0; DE102019116626A1; CN113994246A; EP3987335A1; EP3987335B1

Abstract

In order to check the confocality of a scanning and descanning microscope assembly (3) comprising a light source (4) that provides illumination light (23), comprising an optical unit (27) that focuses the illumination light (23) into a focal region (7) in a focal plane (6), comprising a detector (8) that detects light from the focal region (7) and that has a detection aperture (11) to be arranged in confocal fashion with the focal region (7), and comprising a scanner (12) between the light source (4) and, firstly, the detector (8) and, secondly, the focal plane (6), a detection auxiliary aperture (25), arranged in the focal plane (6), of an auxiliary detector (24) is scanned with the focal region (7) of the illumination light (23) by adjusting the scanner (12), wherein a first intensity distribution of the illumination light (23) registered by the auxiliary detector (24) is captured over different positions of the scanner (12). Furthermore, the detection aperture (11) of the detector (8) is scanned with auxiliary light (22) by adjusting the scanner (12), said auxiliary light emerging through an emission auxiliary aperture (21), arranged in concentric fashion with respect to the detection auxiliary aperture (25) in the focal plane (6), of an auxiliary light source (20), wherein a second intensity distribution of the auxiliary light (22) registered by the detector (8) is captured over the positions of the scanner (12). At least one difference between the first intensity distribution and the second intensity distribution over the different positions of the scanner (12) is captured as a measure for an error of the confocality.

Description

METHODS AND DEVICES FOR CHECKING THE CONFOCALITY OF A SCANNING AND DESCANNING MICROSCOPE ASSEMBLY

TECHNICAL FIELD OF THE INVENTION

The invention relates to a method for checking the confocality of a scanning and descanning microscope assembly with a light source providing illumination light, optics that focus the illumination light into a focus area in a focal plane, a detector that detects light from the focus area, and a detection aperture to be confocally to the focus area has, and a scanner between the light source and the detector on the one hand and the focal plane on the other hand. The invention further relates to a device and to a scanning and descanning microscope assembly for carrying out such a method and to a laser scanning microscope with such a scanning and descanning microscope assembly. Accordingly, the scanning and descanning microscope assembly is, in particular, one for a laser scanning microscope. The detection aperture of the detector can be limited in particular by a pinhole diaphragm arranged in front of the detector and then confocal to the focus area. The detection aperture can, however, also be defined by one or more light-sensitive areas of the detector.

In a scanning and descanning microscope assembly with a light source providing illumination light, optics that focus the illumination light into a focal area in a focal plane, a detector for light from the focal area, which has a detection aperture to be confocally to the focal area, and a scanner between the light source and the Detector with the aperture on the one hand and the focal plane on the other, so that the scanner can scan with the focused illuminating light from the Light source on the one hand and for descanning the light from the focus area to the detector with the pinhole on the other hand, a precisely confocal arrangement of the detection aperture is important in order to optimally measure the light produced by the illuminating light. The precisely confocal arrangement of the detection aperture means that, when it is imaged in the focal plane, it is arranged concentrically to the focal area into which the illuminating light is focused.

STATE OF THE ART

A method for adjusting a laser scanning fluorescence microscope and a corresponding laser scanning fluorescence microscope with an automatic adjustment device are known from WO 2018/042056 A1. This involves setting a correct adjustment of the laser scanning fluorescence microscope, in which an intensity maximum of excitation light and an image of a pinhole diaphragm arranged in front of a fluorescence light detector in the focus of an objective coincide. In the known method, a fluorescent dye-labeled structure in a sample is scanned with the maximum intensity of the excitation light in order to generate images of the sample with images of the structure that correspond to the various apertures of the pinhole. An offset between the positions of the images of the structure in the generated images is then calculated. To set the correct adjustment of the laser scanning fluorescence microscope, the maximum intensity of the excitation light is shifted relative to the image of the pinhole in the direction of the offset, which the image of the structure in an image that corresponds to a smaller aperture of the pinhole, compared to the image of the structure in another image that corresponds to a larger aperture of the pinhole. The automatic adjustment device of the known laser scanning fluorescence microscope is designed to carry out the known method automatically. The known method requires the presence of the complete laser scanning fluorescence microscope and the structure marked with the fluorescent dye in the sample in order to generate the images of the sample with the images of the structure. In addition, the aperture of the pinhole must be variable or at least the pinhole must be removable in order to generate the images that correspond to the different apertures of the pinhole. DE 10 2005 020 542 1 discloses a device and method for setting the position and / or size of a perforated diaphragm in a laser scanning microscope. The pinhole is illuminated via a separate light source or the laser of the laser scanning microscope and the pinhole is displaced transversely to the optical axis until an intensity maximum is present on the detector of the laser scanning microscope located behind the pinhole. The opening of the pinhole is then located at a point which is defined by the optical structure of the laser scanning microscope in relation to the separate light source or the laser and which can be found reproducibly. A confocality of the laser scanning microscope cannot therefore be checked or set. In the laser scanning microscope, the known device is arranged completely on the same side of the scanner as the laser and the detector of the laser scanning microscope.

From WO 2011/047365 A1 an autofocus device with a light source, a beam splitter, a fiber optic circulator, an optical collimator, a comparative detector and a microprocessor is known. The beam splitter splits light from the light source into two parts. The fiber optic circulator has a first connection to the beam splitter, a second connection to the optical collimator and a third connection to the comparative detector. The optical collimator directs some of the light from the fiber optic circulator onto a sample via a dichroic mirror and a microscope objective. The comparative detector uses the other part of the light from the light source as a reference and converts light from a substrate on which the sample is placed into an analog voltage signal. Based on the analog voltage signal from the comparison detector, the microprocessor controls a microscope sample table. In the fiber optic circulator, light is passed only from the first input to the second input and from the second input to the third input. The comparative detector divides the signal reflected from the substrate by the portion of the light from the light source to compensate for variations in the intensity of light from the light source. The microprocessor controls the microscope sample table with the aim of maximizing or keeping the analog voltage signal from the comparison detector to a maximum.

A method and a device for calibrating a confocal laser scanning microscope with a light source providing illumination light, optics that focus the illumination light in a focal area in a focal plane, a light from the focal area Detecting detector, which has a detection aperture to be confocal to the focus area, and a scanner between the light source and the detector on the one hand and the focal plane on the other hand, in which the illuminating light via the scanner to an auxiliary detector and auxiliary light from an auxiliary light source via the scanner to the detector is directed, but without the calibration affecting the confocality of a scanning and descanning microscope assembly are known from DE 199 06 763 A1. The laser scanning microscope scans an object with a light beam. For the calibration, calibration means are arranged in a plane of an intermediate image and can also be scanned with the light beam. The calibration means can be measuring means which are designed as detection means and are used for laser power measurement or laser calibration. The detection means can be photodiodes. The calibration means can also be illuminants which are used to calibrate the detectors. The lamps can be light-emitting diodes. The calibration means can be pivoted into the area of the image field in the plane of the intermediate image or can be fixedly arranged at the edge of the intermediate image outside the actual image field.

OBJECT OF THE INVENTION

The invention is based on the object of providing a method and devices which enable the confocality of a scanning and descanning microscope assembly to be checked without a complete laser scanning microscope comprising the microscope assembly having to be present or a sample having to be measured with such a laser scanning microscope.

SOLUTION

The object of the invention is achieved by a method with the features of independent claim 1, a device with the features of claim 11, a scanning and descanning microscope assembly with the features of claim 12 and a laser scanning microscope with the features of claim 20. The further dependent claims relate to preferred embodiments of the method, the device, the microscope assembly and the laser scanning microscope. DESCRIPTION OF THE INVENTION

The method according to the invention is used to check the confocality of a scanning and descanning microscope assembly with a light source providing illuminating light, the illuminating light emerging from an emission aperture of the light source, an optical system that focuses the illuminating light into a focal area in a focal plane, a detector that detects light from the focal area has a detection aperture to be arranged confocally to the focal area, and a scanner between the light source and the detector with the pinhole on the one hand, d. H. on one side of the scanner and the focal plane on the other hand, d. H. on the other side of the scanner. To this end, it should be noted that the light that is detected with the detector does not have to be emitted from the focal area in the focal plane under consideration, but can also come from another plane. The fact that the detection aperture of the detector is “to be arranged” confocally to the focus area does not define a mandatory step of the method according to the invention, but only that the detection aperture is arranged confocally to the focus area given the confocality of the scanning and descanning microscope assembly of the detector.

In the method according to the invention, an auxiliary detector with an auxiliary detection aperture and / or an auxiliary light source providing auxiliary light with an auxiliary emission aperture from which the auxiliary light emerges is / is arranged in the focal plane. At least the auxiliary detector or the auxiliary light source is arranged in the focal plane. If the auxiliary detector and the auxiliary light source are arranged there, the auxiliary detection aperture and the auxiliary emission aperture are arranged concentrically in the focal plane. To fulfill this feature of the method according to the invention, it may be necessary to actively arrange the auxiliary detector and / or the auxiliary light source in the focal plane. However, it is also fulfilled when the scanning and descanning microscope assembly to be checked for confocality already has the auxiliary detector and / or the auxiliary light source in the focal plane.

In the method according to the invention, the auxiliary detection aperture of the auxiliary detector arranged in the focal plane is scanned by adjusting the scanner with the focus area of the illuminating light, a first intensity distribution of the illuminating light registered by the auxiliary detector being recorded across different positions of the scanner. Alternatively, an auxiliary detection aperture, arranged concentrically to the emission aperture of the light source, of a separate auxiliary detector is created by adjusting the scanner with the auxiliary light, that through the auxiliary emission aperture of the auxiliary light source arranged in the focal plane exits, scanned, a first intensity distribution of the illuminating light registered by the separate auxiliary detector being detected over different positions of the scanner. In both cases, the first intensity distribution describes the arrangement of the emission aperture of the light source and thus of the illuminating light with respect to the auxiliary device arranged in the focal plane, that is, the auxiliary detector or the auxiliary light source.

Furthermore, the detection aperture of the detector is scanned by adjusting the scanner with the auxiliary light that emerges through the auxiliary emission aperture of the auxiliary light source arranged in the focal plane, a second intensity distribution of the auxiliary light registered by the detector being recorded across the positions of the scanner. Alternatively, the auxiliary detection aperture arranged in the focal plane of the auxiliary detector is scanned by adjusting the scanner with auxiliary light which exits through an auxiliary emission aperture of a separate auxiliary light source arranged concentrically to the detection aperture, with a second intensity distribution of the auxiliary light registered by the auxiliary detector across the positions of the scanner is captured. In both cases, the second intensity distribution describes the arrangement of the detection aperture of the detector with respect to the auxiliary device arranged in the focal plane, that is, the auxiliary light source or the auxiliary detector.

At least one difference between the first intensity distribution and the second intensity distribution over the various positions of the scanner is then recorded as a measure of an error in confocality. It goes without saying that the two intensity distributions for detecting the difference are detected over comparable and typically the same positions of the scanner. Then a difference in position between maxima or focal points of the two intensity distributions based on these positions of the scanner indicates an error in the confocality of the microscope assembly, i.e. an offset between the focus area in which the illuminating light is focused into the focus plane with the focusing optics, and the image of the detection aperture of the detector in the focal plane or the image of the focal area into which the illuminating light is focused with the focusing optics onto the detection aperture. Correspondingly, the at least one difference that is preferably detected in the method according to the invention is the difference in position between maxima or focal points of the two intensity distributions. To produce the confocality of the microscope assembly, the at least one difference is compensated for. This can be done by a real relative shift of the detection aperture of the detector with respect to the emission aperture of the light source, which can be achieved by shifting a pinhole in front of the detector or by shifting the light source with respect to the detector, or by a virtual relative shift. A virtual relative displacement is brought about, for example, by pivoting a mirror or by some other movement of another optical component in the beam path of the illuminating light from the light source or the light from the focus area to the detector.

If the method according to the invention is carried out on a laser scanning microscope comprising the scanning and descanning microscope assembly and an objective, the auxiliary detection aperture of the auxiliary detector and / or the auxiliary emission aperture of the auxiliary light source can be arranged in an intermediate image plane of the laser scanning microscope. They can be arranged in an area of the respective intermediate image plane that lies outside the area that is used when an object is scanned with the illuminating light through the objective of the laser scanning microscope. Alternatively, the

Auxiliary detection aperture of the auxiliary detector and / or the auxiliary emission aperture of the auxiliary light source are arranged in a branch from a main beam path of a laser scanning microscope comprising the scanning and descanning microscope assembly and an objective, this branch also generally not leading to the object examined with the laser scanning microscope. This branch can specifically be connected to a beam splitter in

Main beam path of the laser scanning microscope, can be branched off on a deflecting mirror which is arranged stationary or unsteady in the main beam path, or on a rotating mirror of the scanner. The scanners of different laser scanning microscopes have three or four pivotable rotating mirrors, with one or both lateral scanning directions being assigned two rotating mirrors. One of these rotating mirrors per scanning direction can be used to basically align the scanner with the detection auxiliary aperture and / or the emission auxiliary aperture. The respective other mirror can be used to scan the detection auxiliary aperture of the respective auxiliary detector or the detection aperture of the detector. However, many scanners with only one rotating mirror per scanning direction can also be used to scan the auxiliary detection aperture of the auxiliary detector in the branch from the branch from the main beam path of the laser scanning microscope and to scan the detection aperture of the detector with the auxiliary light from the auxiliary light source in the main beam path. So if a suitable scanner is available, it is sufficient to implement the invention In the simplest case, the method comprises a photoelectric component that can be used both as an auxiliary light source and as an auxiliary detector.

In addition, at least some of the optics that focus the illuminating light in the focal area in the focal plane can be arranged in the branch from the main beam path of the laser scanning microscope. In this context, it should be noted that if the auxiliary detection aperture of the auxiliary detector and the auxiliary emission aperture of the auxiliary light source are arranged concentrically in the focal plane in the method according to the invention, this does not necessarily mean that the auxiliary light source and the auxiliary detector actually have to be arranged in the same focal plane. Rather, the auxiliary detection aperture and / or the auxiliary emission aperture can also be arranged in the focal plane in that the auxiliary light source and / or the auxiliary detector are / is imaged there. In addition, the focal plane in which the detection auxiliary aperture and / or the emission auxiliary aperture are arranged does not have to be identical to any intermediate image plane of the laser scanning microscope via which the illuminating light from the light source through the objective to an object to be examined and the light produced by the illuminating light from the object passes through the lens to the detector. Rather, the focal plane in which the detection auxiliary aperture and / or the emission auxiliary aperture are arranged can be offset and / or tilted relative to each intermediate image plane of the laser scanning microscope. In a first embodiment of the method according to the invention, the auxiliary detection aperture of the auxiliary detector arranged in the focal plane is scanned by adjusting the scanner with the focal area of the illuminating light in order to detect the first intensity distribution of the illuminating light registered by the auxiliary detector across the various positions of the scanner. Furthermore, the detection aperture of the detector is scanned by adjusting the scanner with the auxiliary light from the auxiliary light source, which exits through the emission auxiliary aperture of the auxiliary light source arranged concentrically to the detection auxiliary aperture in the focal plane, in order to determine the second intensity distribution of the auxiliary light registered by the detector over the positions of the Across the scanner.

In the first embodiment of the method according to the invention, the detection auxiliary aperture and the emission auxiliary aperture in the focal plane can be not only concentric but also congruent. Coincidence can be brought about, for example, in that the same photoelectric component is used as the auxiliary light source and as the auxiliary detector. Specifically, this photoelectric component can be a light-emitting diode, a Superluminescent diode, a laser diode or a photodiode. The person skilled in the art is generally aware that these photoelectric components can be controlled or connected in such a way that they can be used on the one hand as a light source and on the other hand as a detector for light. In the first embodiment of the method according to the invention, the detection auxiliary aperture and the emission auxiliary aperture can also be formed with an end cross-section of a light guide arranged in the focal plane. This light guide can in principle also lead to a photoelectric component that is used both as an auxiliary light source and as an auxiliary detector. The light guide can, however, also be branched to the auxiliary light source and the auxiliary detector via a free beam beam splitter, a fiber beam splitter or a circulator. Separate photoelectric components can then easily be used for the auxiliary light source and the auxiliary detector. When using a circulator, it is avoided without additional effort that only part of the illuminating light entering the light guide via the end cross section reaches the auxiliary detector or only part of the auxiliary light from the auxiliary light source exits the light guide via the end cross section.

In a second embodiment of the method according to the invention, the auxiliary detection aperture of the auxiliary detector arranged in the focal plane is scanned as in the first embodiment by adjusting the scanner with the focus area of the illumination light in order to obtain the first intensity distribution of the illumination light registered by the auxiliary detector across the various positions of the scanner capture. In contrast to the first embodiment, however, the auxiliary detection aperture arranged in the focal plane of the auxiliary detector is scanned by adjusting the scanner with the auxiliary light that exits through the auxiliary emission aperture of the separate auxiliary light source arranged concentrically to the detection aperture, in order to determine the second intensity distribution of the auxiliary detector registered auxiliary light across the positions of the scanner. In the second embodiment of the method according to the invention, the detection beam path is penetrated by the auxiliary light in a direction which is opposite to the direction of the light normally detected by the detector from the focal plane. Nevertheless, the second intensity distribution describes the arrangement of the detection aperture of the detector - in the form of the arrangement of the concentric emission auxiliary aperture of the auxiliary light source - opposite the auxiliary device - in the form of the auxiliary detector - in the focal plane. In a third embodiment of the method according to the invention, in contrast to the first embodiment, the auxiliary detection aperture of the separate auxiliary detector arranged concentrically to the emission aperture of the light source is scanned by adjusting the scanner with the auxiliary light that exits through the auxiliary emission aperture of the auxiliary light source arranged in the focal plane, in order to to detect first intensity distribution of the illumination light registered by the auxiliary detector over the various positions of the scanner. On the other hand, as in the first embodiment, the detection aperture of the detector is scanned by adjusting the scanner with the auxiliary light that exits through the auxiliary emission aperture of the auxiliary light source arranged in the focal plane, in order to determine the second intensity distribution of the auxiliary light registered by the detector across the positions of the scanner capture. Here, the first intensity distribution describes the arrangement of the emission aperture of the light source - in the form of the arrangement of the auxiliary detection aperture of the auxiliary light detector which is concentric therewith - opposite the auxiliary device - in the form of the auxiliary light source - in the focal plane. In principle, a fourth embodiment of the method according to the invention is also possible, in which, in contrast to the first embodiment, the auxiliary detection aperture of the separate auxiliary detector arranged concentrically to the emission aperture of the light source is achieved by adjusting the scanner with the auxiliary light, and that by the auxiliary emission aperture of the auxiliary light source arranged in the focal plane exits, is scanned in order to detect the first intensity distribution of the illuminating light registered by the auxiliary detector across the various positions of the scanner, and in which, also different from the first embodiment, the auxiliary detector's detection aperture arranged in the focal plane by adjusting the scanner with the auxiliary light , which exits through the emission auxiliary aperture of the separate auxiliary light source arranged concentrically to the detection aperture, is scanned in order to determine the second intensity distribution of the re to detect registered auxiliary light over the positions of the scanner.

To carry out the first embodiment of the method according to the invention, a device with an auxiliary detector and an auxiliary light source can be used, which has a standardized connection for the scanning and descanning microscope assembly or such a standardized connection for a scanning and descanning

Microscope assembly and an objective comprising laser scanning microscope has such a matching mating connection that a detection auxiliary aperture of the auxiliary detector and a Emission auxiliary aperture of the auxiliary light source, the positions of which are fixed with respect to the mating connection, are arranged in the focal plane of the scanning and descanning microscope assembly when the mating connection is connected to the connection. This device can then be connected to the respective microscope assembly or the respective laser scanning microscope via its or its standardized connection in order to check the microscope assembly and thus possibly also the entire laser scanning microscope for its confocality.

In the extreme case, the device according to the invention consists exclusively of the counter connection and an electro-optical component arranged in a fixed position relative to it, which can be used both as an auxiliary detector and as an auxiliary light source, and the associated circuit for controlling this component as an auxiliary detector on the one hand and an auxiliary light source on the other.

When carrying out all embodiments of the method according to the invention, the auxiliary detector and the auxiliary light source can be integrated into the scanning and descanning microscope assembly. A corresponding scanning and descanning microscope assembly according to the invention for carrying out the method according to the invention has a light source providing illuminating light, the illuminating light emerging from an emission aperture of the light source, optics focusing the illuminating light into a focal area in a focal plane, a detector for light from the focal area, which has a Has detection aperture to be arranged confocally to the focus area, a scanner between the light source and the detector on the one hand and the focal plane on the other hand, and in the focal plane an auxiliary detector with an auxiliary detection aperture and / or an auxiliary light source providing auxiliary light with an auxiliary emission aperture from which the auxiliary light emerges. In this case, if the auxiliary detector and the auxiliary light source are arranged in the focal plane, the auxiliary detection aperture and the auxiliary emission aperture are arranged concentrically. By adjusting the scanner, the auxiliary detection aperture of the auxiliary detector can be scanned in the focal plane with the focus area of the illuminating light or an auxiliary detection aperture of a separate auxiliary detector arranged concentrically to the emission aperture of the light source can be scanned with the auxiliary light that exits through the auxiliary emission aperture of the auxiliary light source arranged in the focal plane. Furthermore, by adjusting the scanner, the detection aperture of the detector with the auxiliary light, which emerges in the focal plane from the emission auxiliary aperture of the auxiliary light source, or the detection auxiliary aperture of the auxiliary detector with auxiliary light arranged in the focal plane, which exits through an emission auxiliary aperture of a separate auxiliary light source arranged concentrically to the detection aperture, can be scanned.

Preferably, a microscope assembly according to the invention has a checking device which is designed to adjust the auxiliary detection aperture of the auxiliary detector in the focal plane with the focus area of the illuminating light or the auxiliary detection aperture of the separate auxiliary detector arranged concentrically to the emission aperture of the light source with the auxiliary light from the auxiliary light source in the focal plane and to detect a first intensity distribution of the illumination light registered by the auxiliary detector or of the auxiliary light registered by the separate auxiliary detector over different positions of the scanner, which is furthermore designed to adjust the detection aperture of the detector with the auxiliary light from the auxiliary light source in the focal plane or the auxiliary detection aperture of the auxiliary detector arranged in the focal plane with the one from the concentric to the detection aperture of the detector To scan rdneten emission auxiliary aperture of the separate auxiliary light source emerging auxiliary light and to detect a second intensity distribution of the auxiliary light registered by the detector or the auxiliary light registered by the auxiliary detector across the positions of the scanner, and which is also designed to detect at least one difference between the first intensity distribution and the to detect second intensity distribution across the various positions of the scanner as a measure of an error in the confocality of the microscope assembly. In a scanning and descanning microscope assembly according to the invention for performing the first embodiment of the method according to the invention with the light source providing the illuminating light, the optics focusing the illuminating light into the focal area in the focal plane, the detector for the light from the focal area, which is to be confocally to the focal area Has detection aperture, and the scanner between the light source and the detector on the one hand and the focal plane on the other hand, the auxiliary detection aperture of the auxiliary detector, to which the illuminating light can be directed via the scanner, and the auxiliary emission aperture of the auxiliary light source through which the auxiliary light exits from the auxiliary light source via the scanner can be directed onto the detection aperture of the detector, arranged concentrically in the focal plane, in such a way that the auxiliary detection aperture scans with the focus area of the illuminating light by adjusting the scanner ar and the detection aperture of the detector can be scanned by adjusting the scanner with the auxiliary light from the auxiliary light source. For this purpose, the scanner is between the light source and the detector on the one hand, so that they are on one side of the scanner, and on the other hand, the auxiliary detector and the auxiliary light source, so that they are on the other side of the scanner. Here, too, it should be pointed out again that this arrangement can otherwise be real or virtual, as has already been explained above in connection with the method according to the invention.

A scanning and descanning microscope assembly according to the invention for performing the second embodiment of the method according to the invention has the light source providing the illuminating light, the optics focusing the illuminating light into the focal area in the focal plane, the detector for the light from the focal area, the detection aperture to be confocal to the focal area has, the scanner between the light source and the detector on the one hand and the focal plane on the other hand, and the auxiliary detector to which the illuminating light can be directed via the scanner. The auxiliary detection aperture of the auxiliary detector is arranged in the focal plane, and the auxiliary emission aperture of the separate auxiliary light source providing the auxiliary light is arranged concentrically to the detection aperture of the detector that the auxiliary detection aperture of the auxiliary detector by adjusting the scanner on the one hand with the focus area of the illuminating light and on the other can be scanned with the auxiliary light emerging from the auxiliary emission aperture of the auxiliary light source.

A scanning and descanning microscope assembly according to the invention for carrying out the third embodiment of the method according to the invention has the light source providing the illuminating light, the illuminating light emerging from the emission aperture of the light source, the optics focusing the illuminating light into the focus area in the focal plane, the detector for the light from the Focus area, which has the detection aperture to be confocally to the focus area, the scanner between the light source and the detector on the one hand and the focal plane on the other hand, and the auxiliary light source providing the auxiliary light whose auxiliary light can be directed to the detector via the scanner. The auxiliary emission aperture of the auxiliary light source through which the auxiliary light emerges is arranged in the focal plane, and the auxiliary detection aperture of the separate auxiliary detector is arranged concentrically to the emission aperture of the light source that on the one hand the detection auxiliary aperture of the separate auxiliary detector and on the other hand the detection aperture of the detector can be scanned by adjusting the scanner with the auxiliary light from the auxiliary light source. A scanning and descanning microscope assembly according to the invention for performing the fourth embodiment of the method according to the invention has the light source providing the illuminating light, the illuminating light emerging from the emission aperture of the light source, the optics focusing the illuminating light into the focal area in the focal plane, the detector for the light from the Focal area, which has the detection aperture to be confocally to the focal area, the scanner between the light source and the detector on the one hand and the focal plane on the other hand, the auxiliary detector to which the illuminating light can be directed via the scanner, and the auxiliary light source providing the auxiliary light, whose auxiliary light via the scanner can be aimed at the detector. The auxiliary detection aperture of the auxiliary detector and the auxiliary emission aperture of the auxiliary light source are arranged concentrically in the focal plane in such a way that the auxiliary emission aperture of the separate auxiliary light source providing the auxiliary light is arranged so concentrically to the detection aperture of the detector and the detection auxiliary aperture of the separate auxiliary detector of the light source is arranged concentrically to the emission aperture that the auxiliary detection aperture of the auxiliary detector can be scanned by adjusting the scanner with the auxiliary light emerging from the auxiliary emission aperture of the auxiliary light source and that the auxiliary detection aperture of the separate auxiliary detector can be scanned by adjusting the scanner with the auxiliary light from the auxiliary light source. Preferred embodiments of the microscope assembly according to the invention have already been explained in connection with the method according to the invention. In a laser scanning microscope according to the invention with an objective and a scanning and descanning microscope assembly according to the invention, the auxiliary detection aperture of the auxiliary detector and / or the auxiliary emission aperture of the auxiliary light source can be arranged in an intermediate image plane of the laser scanning microscope or in a branch from a main beam path of the laser scanning microscope. This branch can optionally branch off at a beam splitter in the main beam path of the laser scanning microscope, at a deflecting mirror or a rotating mirror of the scanner. As an alternative or in addition, at least part of the optics that focus the illuminating light into the focal area in the focal plane can be arranged in the junction.

The detection aperture of the detector can be used in any embodiment of the scanning and descanning microscope assembly according to the invention and the microscope assembly according to the invention

Laser scanning microscope can be delimited by a perforated diaphragm arranged in front of the detector, ie its light-sensitive area. In any embodiment of the scanning and descanning microscope assembly according to the invention and of the laser scanning microscope according to the invention, the detector can be a point detector or an array detector. The array detector can have a small field of 2x2 to 10x10 light-sensitive elements or it can, for example, have 13 light-sensitive elements arranged on a hexagonal grid in order to register the Airy disk of the light imaged on the detector from the focus area of the focal plane in a spatially resolved manner. In the case of an array detector, the second intensity distribution of the auxiliary light registered by the detector can be the sum of the individual intensity distributions registered by all or some of its individual elements across the positions of the scanner, or also just a selected individual one of these individual intensity distributions. It is also possible to determine the positions of the individual elements of the array detector relative to the auxiliary emission aperture of the auxiliary light source in the focal plane from the differences between the individual intensity distributions.

In principle, each auxiliary detector can also be not only a point detector but also an array detector.

Advantageous developments of the invention emerge from the claims, the description and the drawings. The advantages of features and of combinations of several features mentioned in the description are only exemplary and can come into effect alternatively or cumulatively without the advantages necessarily having to be achieved by embodiments according to the invention. Without changing the subject matter of the appended claims, the following applies with regard to the disclosure content of the original application documents and the patent: Further features are the drawings - in particular the geometries shown and the relative dimensions of several components to one another and their relative arrangement and operative connection - refer to. The combination of features of different embodiments of the invention or of features of different claims is also possible in a way deviating from the selected back-references of the claims and is hereby suggested. This also applies to features that are shown in separate drawings or mentioned in their description. These features can also be combined with features of different patent claims. Features listed in the claims for further embodiments of the invention can also be omitted. The number of features mentioned in the claims and the description are to be understood in such a way that precisely this number or a greater number than the stated number is present without the need for the explicit use of the adverb "at least". If, for example, a deflection mirror is mentioned, this is to be understood to mean that precisely one deflection mirror, two deflection mirrors or more deflection mirrors are present. The features cited in the claims can be supplemented by other features or be the only features that the respective method or the respective product has.

The reference signs contained in the patent claims do not restrict the scope of the subject matter protected by the patent claims. They only serve the purpose of making the patent claims easier to understand.

BRIEF DESCRIPTION OF THE FIGURES

In the following, the invention is further explained and described with reference to preferred embodiment examples shown in the figures. 1 shows a laser scanning microscope according to the invention with a first embodiment of the microscope assembly according to the invention when performing a step of several embodiments of the method according to the invention.

FIG. 2 shows the laser scanning microscope according to the invention according to FIG. 1 in FIG

Carrying out a further step of several embodiments of the method according to the invention.

Fig. 3 shows a further embodiment of the laser scanning microscope according to the invention with a second embodiment of the microscope assembly according to the invention.

Fig. 4 shows a scanning and descanning microscope assembly with a connected device according to the invention. Fig. 5 shows a further embodiment of the laser scanning microscope according to the invention with a third embodiment of the microscope assembly according to the invention.

Fig. 6 shows a detail of a fourth embodiment of the microscope assembly according to the invention.

Fig. 7 is a circuit diagram for the control of a light-emitting diode, the various

Embodiments of the microscope assembly according to the invention and the device according to the invention can be used both as an auxiliary detector and as an auxiliary light source, and FIGS. 8 to 10 illustrate a first, a second and a third embodiment of the method according to the invention on microscope assemblies according to the invention that are adapted but only shown schematically.

FIGURE DESCRIPTION

The laser scanning microscope 1 shown schematically in FIG. 1 comprises an objective 2 and a microscope assembly 3, the components of which are outlined with a dashed line. The microscope assembly 3 includes a light source 4, in particular in the form of a laser 5, for providing illuminating light. In the step of the method according to the invention illustrated in FIG. 1, however, the light source 4 is not activated in order to provide the illuminating light. Furthermore, the microscope assembly 3 includes optics that focus the illuminating light in a focal area 7 in a focal plane 6. In FIG. 1, the focal plane 6 is shown with a dashed line and the focus area 7 in the focus plane 6 is shown with a point. The components of the focusing optics are not shown. The microscope assembly 3 also includes a detector 8 for light 9 from the focal area 7. In front of the actual detector 8, ie its light-sensitive area, there is a perforated diaphragm 10. The opening of the perforated diaphragm 10 defines a detection aperture 11 of the detector 8, which at correct adjustment of the microscope assembly 3 is arranged confocally to the focus area 7. Between the light source 4 and the detector 8 with the perforated diaphragm 10 on the one hand and the focal plane 6 on the other hand, a scanner 12 of the microscope assembly 3, which is shown only schematically in FIG. 1, is arranged. The scanner comprises at least one tilting mirror 13, 14 per lateral direction, in which the scanner is provided for scanning a sample 15 arranged in front of the objective 2 with the illuminating light from the light source 4. However, the scanner 12 not only serves to scan the sample 15, but also to descan the light 9 that is caused by the illuminating light in the sample 15, so that this light is selectively detected by the detector 8. What is important here is an exact confocality of the microscope assembly 3, ie an exact coincidence of the focal area 7 of the illumination light focused into the sample 15 and an image of the opening 11 of the detection aperture 11 of the detector 8 in the sample 15. This confocality is checked in the method according to the invention in the focal plane 6 shown in FIG. 1. This focal plane 6 lies here in a branch 16 from the main beam path 17 of the laser scanning microscope. The branch 16 branches off from the main beam path 17 at a beam splitter 18. In the branch 16, a photoelectric component 19 is arranged, which is used as an auxiliary light source 20 in the step of the method according to the invention, which is illustrated in FIG. 1. The auxiliary light source 20 has an emission auxiliary aperture 21 in the focal plane 6. In the step according to FIG. 1, by adjusting the scanner 12, the detection aperture 11 of the detector 8 is scanned with auxiliary light 22 from the auxiliary light source 20, which exits through the auxiliary emission aperture 21 in the focal plane 6. In this case, an intensity distribution of the auxiliary light 22 registered by the detector 8 is recorded over the various positions of the scanner 12, ie assigned to these various positions of the scanner 12. In other words, a scan image of the detection aperture 11 of the detector 8 is recorded with the aid of the auxiliary light source 20.

In the step of the method according to the invention, which is illustrated in FIG. 2, the light source 4 is activated to provide the illuminating light 23, and the photoelectric component 19 is used as an auxiliary detector 24. Then a detection auxiliary aperture 25 of the auxiliary detector 24, which is located in the focal plane 6 and is arranged concentrically to the emission auxiliary aperture 21 according to FIG. 1, is scanned with the illuminating light 23, with a further intensity distribution of the illuminating light 23 registered by the auxiliary detector 24 over the various positions of the Scanner 12 is detected away. In this step, a scan image of the auxiliary detection aperture 25 of the auxiliary detector 24 is recorded in the focal plane 6. The scan image of the detection aperture 11 of the detector 8, which is recorded according to FIG. 1, and the scan image of the detection auxiliary aperture 25, which is recorded according to FIG. 2, are concentric with respect to the associated positions of the scanner 12 when the opening of the pinhole 10 is arranged exactly confocal to the focus area 7 in the focal plane 6 in which the illuminating light 23 is focused. A concentricity of the two Scan images means in particular that the maxima of the two intensity distributions and / or their focal points coincide. Every ascertainable difference in position between the maxima or the focal points indicates an error in the confocality of the microscope assembly 3, which is to be compensated for by a real or virtual relative displacement of the light source 4 with respect to the detector 8 or its detection aperture 11 in order to achieve the

Adjust the microscope assembly 3 correctly. A virtual relative displacement can be effected, for example, by tilting a beam splitter 26 on the side of the scanner 12 facing away from the objective 2, which merges or separates the beam paths of the illuminating light 23 from the light source 4 and the light 9 to the detector 8. In the reproduction of the laser scanning microscope 1 according to the invention in FIG. 3, the optics 27, which focus the illuminating light in the focal area 7 in the focal plane 6, are shown. These optics 27 include a so-called scan lens 28. Another optics 29 focuses the scanned light 9 to be detected by the detector 8 onto the opening of the perforated diaphragm 10, i. H. into the detection aperture 11 of the detector 8. The photoelectric component 19 is imaged into the focal plane 6 with yet another optics 30 in order to virtually arrange the auxiliary detection aperture 25 and the auxiliary emission aperture 21 in the focal plane 6. Alternatively, the optics 30 can be viewed as part of the optics 27 and, accordingly, the focal plane 6 drawn in FIG. 3 as an intermediate focal plane and the further focal plane 31 drawn there, in which the photoelectric component 19 is arranged, as the focal plane in which the emission auxiliary aperture 21 the auxiliary light source 20 then real concentric to the

Detection auxiliary aperture 25 of the auxiliary detector 24 is arranged. Quite generally, the optics 27 can have one or more intermediate focus planes between the light source 4 and the detector 8 with the pinhole 10 on the one hand and the focal plane 6 in which the photoelectric component 19 is arranged, on the other hand. The focal plane 6 drawn in FIG. 3 in the branch 16 corresponds to an intermediate image plane 32 in the main beam path 17 of the

Scanning microscope 1. A tube lens 33 is arranged in this main beam path 17 between the intermediate image plane 32 and the objective 2. Finally, instead of the beam splitter 18 according to FIGS. 1 and 2, FIG. 3 shows a deflecting mirror 34 which is temporarily introduced into the main beam path 17 to carry out the method according to the invention in order to activate the branch 16 to the focal plane 6.

In Fig. 3 a checking device 61 is sketched, which the steps of the method according to the invention explained with reference to FIGS. 1 and 2 in basically the same way in the 3 and automatically controls the photoelectric component 19, the light source 4 and an actuator for the beam splitter 26, not shown separately, as well as the intensities registered by the detector 8 and the photoelectric component 19 operated as auxiliary detector 24 of the auxiliary light 22 or the illumination light 23 reads. The checking device 61 can be provided in all microscope assemblies 3 according to the invention and also when a microscope assembly according to the prior art is temporarily combined with a device according to the invention in order to carry out the method according to the invention, as will be explained with reference to the following FIG. 4 does not show a complete laser scanning microscope, but only a scanning and descanning assembly 60 for such a laser scanning microscope. This microscope assembly 60 according to FIG. 4 corresponds as such to the prior art. In contrast to FIGS. 1 to 3, the scanner 12 has a total of four rotating mirrors 13 and 14, of which two rotating mirrors 13 and two rotating mirrors 14 are assigned to each of the two deflection directions of the scanner 12. The scanning lens 28 of the optics 27 is located on the side of the scanner 12 facing the light source 4 and detector 8. On the side of the scanner 12 facing away from the light source 4 and detector 8, the microscope assembly 60 is attached to a housing 35 or another supporting structure a connection 36 is designed to align the microscope assembly 60 with respect to a corresponding connection, in particular a camera connection of a light microscope with the objective 2 according to FIGS. 1 to 3, and to mount it thereon. According to FIG. 4, however, a device 37 according to the invention with a mating connection 38 matching the connection 36 is mounted on the connection 36 such that an end cross section 39 of a light guide 40 is arranged in the focal plane 6, into which the optics 27 focus the illuminating light 23. The light guide 40 leads via a fiber optic circulator 41 to a photodiode 42 serving as an auxiliary detector 24. A laser diode 43 is connected to the light guide 40 as an auxiliary light source 20 via the same circulator 41. The circulator 41 exclusively forwards the illuminating light 23 entering the end cross section 39 of the light guide 40 to the auxiliary detector 24, while it guides the auxiliary light 22 from the auxiliary light source 20 exclusively to the end cross section 39 of the light guide 40. The end cross-section 39 of the light guide 40 forms the detection auxiliary aperture 25 and the emission auxiliary aperture 21 in the focal plane 6. This focal plane 6 is located in the main beam path 17 according to FIG. 4. The method according to the invention can be carried out using the device 37 in order to confocal the microscope assembly 60 Arrangement of the opening 11 of the aperture plate 10 to the To check the focus area 7, in which the illuminating light 23 is focused in the focal plane 6, as well as to determine any confocality errors and then to correct them by moving the aperture plate 10 relative to the light source 4.

If the auxiliary detector 24 is a separate component from the auxiliary light source 20 as shown in FIG. 4, the two steps of the method according to the invention explained with reference to FIGS. 1 and 2 can also be carried out simultaneously, with the detector 8 and the auxiliary detector 24 being the two Intensity distributions, the differences of which indicate an error in the confocality of the microscope assembly 60, are recorded simultaneously. The two intensity distributions are then automatically assigned to the same positions of the scanner 12. In the embodiment of the laser scanning microscope 1 according to the invention according to FIG. 5, the microscope assembly 3 basically corresponds to that according to FIG. 4. However, it is a microscope assembly according to the invention with an integrated photoelectric component 19 which is used as an auxiliary light source 20 and auxiliary detector 24. Unlike in FIGS. 1 to 3, however, no additional beam splitter 18 or deflecting mirror 34 is arranged in the main beam path 17 in order to deflect the illuminating light 23 to the auxiliary detector 24 arranged in the branch 16 or to supply the auxiliary light 22 from the auxiliary light source 20 to steer the detector 8, but for this purpose a rotary mirror 14 of the four rotary mirrors 13, 14 is used, which are shown here together with their rotary drives 44. The other rotating mirrors 13, 14 can then be used to scan the auxiliary detection aperture 25 of the auxiliary detector 24 or the detection aperture 11 of the detector 8. The photoelectric component 19 is located opposite the other components of the microscope assembly 3 in a stationary position in which the detection auxiliary aperture 25 and the emission auxiliary aperture 21 lie in the focal plane 6 and in which the photoelectric component 19 with the illuminating light 23 can be reached via the scanner 12. In the detail of a further microscope assembly according to the invention shown in FIG. 6, the branch 16 branches off in an edge region 45 of a scanning lens 28 of the focusing optics 27. The illuminating light 23 passing through this edge area 45 is deflected by a deflecting mirror 46 in the form of an edge mirror 47 to the photoelectric component 19, whereby it passes through a further lens 48 of the focusing optics 27 and is thus focused into the focal plane 6. In this way, a very compact arrangement of the photoelectric component 19, which includes both the auxiliary light source 20 and the auxiliary detector 24 forms, reached in the microscope assembly 3. 6 further explains that the auxiliary emission aperture 21 and the auxiliary detection aperture 25 can be specified not only by the auxiliary light source 20 and the auxiliary detector 24, ie here the photoelectric component 19, but also with an additional pinhole arranged in particular in the focal plane 16 62. If the opening of the perforated diaphragm 62 is smaller than the light-emitting area of the auxiliary light source 20 or the light-sensitive area of the auxiliary detector 24, it defines the auxiliary emission aperture 21 and the auxiliary detection aperture 25.

Fig. 7 shows a possible driver circuit 49 for a light emitting diode 50 for use as the photoelectric component 19, i. H. both as an auxiliary light source 20 and as an auxiliary detector 24. In the switching position of two changeover switches 51 and 52 shown, the light-emitting diode 50 is connected to an operational amplifier 53 for use as an auxiliary detector 24. When switching the two switches 51 and 52, the light-emitting diode 50 is operated as an auxiliary light source, the intensity of the auxiliary light via two transistors 54 and 55 being adjustable. The operating mode of the light-emitting diode 50 is selected via a signal input 56. The intensity of the auxiliary light from the LED 50 is selected via control inputs 57 and 58, and the intensity of the illuminating light registered by the light-emitting diode 50 as auxiliary detector 24 is output at a signal output 59.

8 illustrates a first embodiment of the method according to the invention for checking the confocality of the scanning and descanning microscope assembly 1, which is only shown schematically here. The microscope assembly 1 here additionally has a deflecting mirror 64 for deflecting the detection beam path to the detector 8 and further optics 63 in the illumination beam path between the light source 4 and the beam splitter 26. In addition, an emission aperture 65 of the light source 4, from which the illuminating light 23 emerges, is shown, and the electro-optical component 19 is additionally provided with a further reference symbol for an auxiliary device 66 to be arranged in the focal plane 6 to carry out the method according to the invention.

The first embodiment of the method according to the invention comprises the steps as explained with reference to FIGS. 1 and 2. That is, by adjusting the scanner 12 with the aid of the illuminating light 23 coming from the light source 4, the auxiliary emission aperture 25 of the auxiliary detector 24 in the focal plane 6 is scanned, and by adjusting the scanner 12, the auxiliary light source 20 emerging from the auxiliary emission aperture 21 is also scanned Auxiliary light 22 scanned the detection aperture 11 of the detector 8. The two intensity distributions of the illumination light 23 registered by the auxiliary detector 24 for the illumination beam path and the auxiliary light 22 registered by the detector 8 for the detection beam path, recorded over the positions of the scanner 12, are exactly the same, i.e. have the same maxima and / or centers of gravity when the confocality of the microscope assembly 1 is given.

In a second embodiment of the method according to the invention according to FIG. 9, the step according to FIG. 2 is carried out, that is, the detection auxiliary aperture 25 of the auxiliary light source 4 in the focal plane 6 is scanned with the illuminating light 23 from the light source 4 by adjusting the scanner 12, to detect the orientation of the illumination beam path. However, the orientation of the detection beam path is not detected by scanning the detection aperture 11 of the detector 8 with auxiliary light 22 which emerges from an emission auxiliary aperture 21 in the focal plane 6 arranged concentrically to the detection auxiliary aperture 25. Rather, the direction of irradiation of the detection beam path is reversed and the detector 8 is replaced by an auxiliary light source 20 'which is separate from the auxiliary device 66, the emission auxiliary aperture 2 of which, like the detection aperture 11 of the detector 8, is defined by the opening of the aperture plate 10. With the auxiliary light 22 ′ exiting through this auxiliary detection aperture 2, the auxiliary detection aperture 25 of the auxiliary detector 24 is scanned in the focal plane 6 by adjusting the scanner 12. The intensity distribution of the auxiliary light 22 'registered by the auxiliary detector 24 via the position of the scanner is then the second intensity distribution describing the orientation of the detection beam path, which is compared when checking the confocality of the microscope assembly 1 with the first intensity distribution describing the orientation of the illumination beam path. While in the second embodiment of the method according to the invention according to FIG. 9 the auxiliary device 66 in the focal plane 6 is only the auxiliary detector 24 and a separate auxiliary light source 20 'is used at the location of the detector 8, in the third embodiment of the method according to the invention according to FIG. 10 the auxiliary device 66 in the focal plane 6 only the auxiliary light source 20. With the auxiliary light 22 emitted by the auxiliary light source 20 through its auxiliary emission aperture 21, the detection aperture 11 of the detector 8 is scanned in the step according to FIG. 1 by adjusting the scanner 12. Furthermore, in a departure from the procedure according to FIG. 2, the same auxiliary light 22 is used via the same positions of the scanner 12 Scanning an auxiliary detection aperture 25 'of an auxiliary detector 24' separate from auxiliary device 66 at the location of light source 4. The same location of light source 4 and the separate auxiliary detector 24 'can be implemented, for example, by having emission aperture 25 of light source 4 and detection auxiliary aperture 25 'of the auxiliary detector 24' is formed by the same surface of an optical fiber which branches via a circulator to the light source 4 on the one hand and the auxiliary detector 24 'on the other hand. In the third embodiment according to FIG. 10, the direction of the irradiation of the illumination beam path from the light source 4 to the focal plane 6 is reversed for checking the confocality of the microscope assembly 1. The first intensity distribution of the auxiliary light 22 registered with the separate auxiliary detector 24 'thereby corresponds to the first intensity distribution otherwise registered according to FIG. 2 for the illumination beam path and can just as well be compared with the second intensity distribution recorded for the detection beam path in order to determine the confocality of the microscope assembly 1 check.

REFERENCE LIST

Laser scanning microscope

lens

Microscope assembly

Light source

laser

Focus plane

Focus area

detector

light

Pinhole

Detection aperture of the detector 8

scanner

Rotating mirror

sample

Branch

Main beam path

Beam splitter

photoelectric component

Auxiliary light source

separate auxiliary light source

Auxiliary emission aperture of the auxiliary light source 20

Auxiliary emission aperture of the separate auxiliary light source 20 '

Auxiliary light

Illuminating light

Auxiliary detector

'' separate auxiliary detector

Detection auxiliary aperture of the auxiliary detector 24

Detection auxiliary aperture of the separate auxiliary detector 24

Beam splitter

focusing optics

Scanning lens further optics

further optics

Focus plane

Tube lens intermediate image plane

Deflection mirror

casing

connection

contraption

Mating connection end cross-section

Light guide

Circulator

Photodiode

Laser diode

Rotary drive

Edge area

Deflection mirror

Edge mirror

lens

Driver circuit LED

Toggle switch

Operational amplifier transistor

transistor

Signal input

Control input

Signal output

Microscope assembly inspection device pinhole further optics

mirror

Emission aperture of the light source 4 auxiliary device

Claims

PATENT CLAIMS

1. Method for checking the confocality of a scanning and descanning microscope assembly (3) with

a light source (4) providing illumination light (23), the illumination light (23) emerging from an emission aperture (65) of the light source (4),

an optical system (27) that focuses the illuminating light (23) in a focal area (7) in a focal plane (6),

a detector (8) which detects light from the focus area (7) and has a detection aperture (11) to be arranged confocally to the focus area (7),

a scanner (12) between the light source (4) and the detector (8) on the one hand and the focal plane (6) on the other hand, and

wherein an auxiliary detector (24) with an auxiliary detection aperture (25) and / or an auxiliary light source (20) providing auxiliary light (22) with an auxiliary emission aperture (21) from which the auxiliary light (22) emerges is arranged in the focal plane (6) / are, wherein, when the auxiliary detector (24) and the auxiliary light source (20) are arranged in the focal plane (6), the detection auxiliary aperture (25) and the emission auxiliary aperture (21) are arranged concentrically in the focal plane (6), the in the focal plane (6) arranged detection auxiliary aperture (25) of the auxiliary detector (24) by adjusting the scanner (12) with the focus area (7) of the illuminating light (23) is scanned, a first intensity distribution of the illuminating light registered by the auxiliary detector (24) ( 23) is detected across different positions of the scanner (12), or an auxiliary detection aperture (25 ') of a separate auxiliary detector arranged concentrically to the emission aperture (65) of the light source (4) rs (24 ') is scanned by adjusting the scanner (12) with the auxiliary light (22) which emerges through the auxiliary emission aperture (21) of the auxiliary light source (20) arranged in the focal plane (6), with a first intensity distribution of the separate auxiliary detector (24 ') registered illumination light (23) is detected across different positions of the scanner (12), the detection aperture (1 1) of the detector (8) by adjusting the scanner (12) with the auxiliary light (22), the through the emission auxiliary aperture (21) of the auxiliary light source (20) arranged in the focal plane (6), is scanned, a second intensity distribution of the auxiliary light (22) registered by the detector (8) over the positions of the scanner (12) being detected , or the auxiliary detection aperture (25) of the focal plane (6) Auxiliary detector (24) is scanned by adjusting the scanner (12) with auxiliary light (22 ') which emerges through an emission auxiliary aperture (21') of a separate auxiliary light source (20 ') arranged concentrically to the detection aperture (1 1), a second Intensity distribution of the auxiliary light (22) registered by the auxiliary detector (24) is detected over the positions of the scanner (12), and

that at least one difference between the first intensity distribution and the second intensity distribution across the various positions of the scanner (12) is detected as a measure of an error in confocality.

2. The method according to claim 1, characterized in that the at least one difference is a difference in position between maxima or focal points of the first Intensitätsvertei development and the second intensity distribution.

3. The method according to any one of the preceding claims, characterized in that the at least one difference is compensated for by a real and or virtual relative displacement of the detection aperture (1 1) of the detector (8) with respect to the light source (4).

4. The method according to any one of the preceding claims, characterized in that the detection auxiliary aperture (25) and / or the emission auxiliary aperture (21) in the

Focal plane is / are arranged, in an intermediate image plane a laser scanning microscope comprising the scanning and descanning microscope assembly (3) and an objective (2) is / are arranged or

that the detection auxiliary aperture (25) and / or the emission auxiliary aperture (21), which is / are arranged in the focal plane, in a branch from a main beam path of a laser scanning microscope (1) comprising the scanning and descanning microscope assembly (3) and an objective (2) is / are arranged, being optional

the branch (16) is branched off at a beam splitter (18), a deflecting mirror (34) or a rotating mirror (13) of the scanner (12) in the main beam path (17) of the laser scanning microscope (1) and / or

the optics (27) focussing the illuminating light (23) in the focal area (7) in the focal plane (6) is arranged at least partially in the branch (16).

5. The method according to any one of claims 1 to 4 for checking the confocality of the scanning and descanning microscope assembly (3) the light source (4) providing the illuminating light (23),

the optics (27) focusing the illuminating light (23) in a focus area (7) in the focal plane (6),

the detector (8) which detects the light from the focus area (7) and which has the detection aperture (11) to be arranged confocally to the focus area (7), and

the scanner (12) between the light source (4) and the detector (8) on the one hand and the focal plane (6) on the other hand,

wherein the illuminating light (23) is directed via the scanner (12) onto the auxiliary detector (24) and

wherein the auxiliary light (22) is directed from the auxiliary light source (20) via the scanner (12) onto the detector (8),

characterized,

that the auxiliary detection aperture (25) of the auxiliary detector arranged in the focal plane (6)

(24) is scanned by adjusting the scanner (12) with the focus area (7) of the illuminating light (23) in order to determine the first intensity distribution of the illuminating light (23) registered by the auxiliary detector (24) over the various positions of the scanner (12) to capture away, and

that the detection aperture (11) of the detector (8) through the adjustment of the scanner (12) with the auxiliary light (22), which through the emission auxiliary aperture (21) of the auxiliary light source (21) arranged concentrically to the detection auxiliary aperture (25) in the focal plane (6) 20) exits, is scanned in order to detect the second intensity distribution of the auxiliary light (22) registered by the detector (8) over the positions of the scanner (12).

6. The method according to claim 5, characterized in that the detection auxiliary aperture

(25) of the auxiliary detector (24) and the auxiliary emission aperture (21) of the auxiliary light source (20) are congruent in the focal plane (6).

7. The method according to claim 5 or 6, characterized in that the same photoelectric component (19) is used as the auxiliary light source (20) and as the auxiliary detector (24), the photoelectric component (19) optionally a light emitting diode (50), a Superluminescent diode, a laser diode (43) or a photodiode (42) is.

8. The method according to claim 5 or 6, characterized in that the detection auxiliary aperture (25) of the auxiliary detector (24) and the emission auxiliary aperture (21) of the Auxiliary light source (20) with an end cross-section (39) of a light guide (40) arranged in the focal plane (6), the light guide (40) optionally via a free beam beam splitter, a fiber beam splitter or a circulator (41) to the auxiliary light source ( 20) and the auxiliary detector (24) is branched.

9. The method according to any one of claims 1 to 4 for checking the confocality of the scanning and descanning microscope assembly (3)

the light source (4) providing the illuminating light (23),

the optics (27) that focus the illuminating light (23) into the focal area (7) in the focal plane (6),

the detector (8) which detects from the focus area (7) and which has the detection aperture (11) to be arranged confocally to the focus area (7), and

wherein the illuminating light (23) is directed via the scanner (12) onto the auxiliary detector (24),

characterized,

that the auxiliary detection aperture (25) of the auxiliary detector (24) arranged in the focal plane (6) by adjusting the scanner (12) with the focal area (7) of the illuminating light

(23) is scanned in order to detect the first intensity distribution of the illuminating light (23) registered by the auxiliary detector (24) over the various positions of the scanner (12), and

(24) by adjusting the scanner (12) with the auxiliary light (22), which exits through the emission auxiliary aperture (21) of the separate auxiliary light source (20 ') arranged concentrically to the detection aperture (11), is scanned to produce the second intensity distribution of the auxiliary light (22) registered by the auxiliary detector (24) across the positions of the scanner (12).

10. The method according to any one of claims 1 to 4 for checking the confocality of the scanning and descanning microscope assembly (3)

the light source (4) providing the illuminating light (23), the illuminating light (23) emerging from the emission aperture (65) of the light source (4),

the optics (27) which focus the illuminating light (23) in the focal area (7) in the focal plane (6), the detector (8) which detects the light from the focus area (7) and which has the detection aperture (1 1) to be arranged confocally to the focus area (7), and

the scanner (12) between the light source (4) and the detector (8) (10) on the one hand and the focal plane (6) on the other hand,

characterized,

that the detection auxiliary aperture (25) of the separate auxiliary detector (24 '), which is arranged concentrically to the emission aperture (65) of the light source (4), is caused by the adjustment of the scanner (12) with the auxiliary light (22), which through the in the focal plane (6) arranged emission auxiliary aperture (21) of the auxiliary light source (20) exits, is scanned in order to detect the first intensity distribution of the illumination light (23) registered by the auxiliary detector (24) over the various positions of the scanner (12), and

that the detection aperture (1 1) of the detector (8) is scanned by adjusting the scanner (12) with the auxiliary light (22) which exits through the emission auxiliary aperture (21) of the auxiliary light source (20) arranged in the focal plane (6) in order to detect the second intensity distribution of the auxiliary light (22) registered by the detector (8) over the positions of the scanner (12).

1 1. Device (37) for performing the method according to one of claims 5 to 8, with an auxiliary detector (24) and an auxiliary light source (20), characterized by a standardized connection (36) of the scanning and descanning microscope assembly (3) or a laser scanning microscope (1) which includes the scanning and descanning microscope assembly (3) and an objective (2), a mating connection (38) that matches an auxiliary detection aperture (25) of the auxiliary detector (24) and an auxiliary emission aperture (21) of the auxiliary light source (20) whose positions with respect to the mating connection (38) are fixed, are arranged in the focal plane (6) of the scanning and descanning microscope assembly (3) when the mating connection (38) is connected to the connection (36).

12. Scanning and descanning microscope assembly (3) for performing the method according to one of claims 1 to 10 with

a light source (4) providing illumination light (23), the illumination light (23) emerging from an emission aperture (65) of the light source (4), an optical system (27) that focuses the illuminating light (23) in a focal area (7) in a focal plane (6),

a detector (8) for light from the focus area (7) which has a detection aperture (11) to be arranged confocally to the focus area (7),

a scanner (12) between the light source (4) and the detector (8) on the one hand and the focal plane (6) on the other hand,

an auxiliary detector (24) with an auxiliary detection aperture (25) and / or an auxiliary light source (20) providing auxiliary light (22) with an auxiliary emission aperture (21) from which the auxiliary light (22) emerges in the focal plane (6), in which case, when the auxiliary detector (24) and the auxiliary light source (20) are arranged in the focal plane, the auxiliary detection aperture (25) and the auxiliary emission aperture (21) are arranged concentrically,

whereby, by adjusting the scanner (12), the auxiliary detection aperture (25) of the auxiliary detector (24) is arranged in the focal plane (6) with the focal area (7) of the illuminating light (23) or a concentric to the emission aperture (65) of the light source (4) Detection auxiliary aperture (25 ') of a separate auxiliary detector (24') can be scanned with the auxiliary light (22) which emerges through the emission auxiliary aperture (21) of the auxiliary light source (20) arranged in the focal plane (6) and

whereby, by adjusting the scanner (12), the detection aperture (11) of the detector (8) with the auxiliary light (22) which emerges in the focal plane (6) from the emission auxiliary aperture (21) of the auxiliary light source (20), or in the focal plane (6) arranged detection auxiliary aperture (25) of the auxiliary detector (24) with auxiliary light (22 ') which exits through an emission auxiliary aperture (2T) arranged concentrically to the detection aperture (1 1) of a separate auxiliary light source (20') can be scanned.

13. microscope assembly (3) according to claim 12, characterized by a checking device (61) which is designed to

By adjusting the scanner (12), the auxiliary detection aperture (25) of the auxiliary detector (24) in the focal plane (6) with the focal area (7) of the illuminating light (23) or the auxiliary detection aperture arranged concentrically to the emission aperture (65) of the light source (4) (25 ') of the separate auxiliary detector (24') with the auxiliary light (22) from the auxiliary light source (20) in the focal plane (6) and a first intensity distribution of the illumination light (23) registered by the auxiliary detector (24) or that of the separate auxiliary detector (24 ') to detect auxiliary light (22) registered across different positions of the scanner (12), by adjusting the scanner (12) the detection aperture (11) of the detector (8) with the auxiliary light (22) from the auxiliary light source (20) in the focal plane (6) or the auxiliary detector aperture (25) arranged in the focal plane (6) (24) with the auxiliary light (22 ') emerging from the concentric to the detection aperture (11) of the detector (8) arranged emission auxiliary aperture (2T) of the separate auxiliary light source (20') and a second intensity distribution of the recorded by the detector (8) Auxiliary light (22) or the auxiliary light (22 ') registered by the auxiliary detector (24) across the positions of the scanner (12), and

to detect at least one difference between the first intensity distribution and the second intensity distribution over the various positions of the scanner (12) as a measure of an error in confocality.

14. microscope assembly (3) according to claim 13, characterized in that the checking device (61) is designed to detect the at least one difference by a real and / or virtual relative displacement of the detection aperture (1 1) of the detector (8) with respect to the light source ( 4) to automatically compensate.

15. microscope assembly (3) according to any one of claims 13 and 14, characterized in that the detection auxiliary aperture (25, 25 ') of the auxiliary detector (24) and the separate auxiliary detector (24') and the emission auxiliary aperture (21, 2T) of the Auxiliary light source (20) and the separate auxiliary light source (20 '), and optionally, if available, the auxiliary light source (20) and the separate auxiliary light source (20') as well as the auxiliary detector (24) and the separate auxiliary detector (24 '), in a housing of the microscope assembly (3) are arranged.

16. microscope assembly (3) according to any one of claims 12 to 15 for performing the method according to any one of claims 5 to 8 with

the light source (4) providing the illuminating light (23),

the detector (8) for the light from the focus area (7), which has the detection aperture (11) to be arranged confocally to the focus area (7),

the scanner (12) between the light source (4) and the detector (8) on the one hand and the focal plane (6) on the other hand, the auxiliary detector (24) to which the illuminating light (23) can be directed via the scanner (12), and

the auxiliary light source (20) providing the auxiliary light (22), the auxiliary light (22) of which can be directed onto the detector (8) via the scanner (12),

characterized in that the auxiliary detection aperture (25) of the auxiliary detector (24) and the auxiliary emission aperture (21) through which the auxiliary light (22) emerges from the auxiliary light source (20) are arranged concentrically in the focal plane (6) that the auxiliary detection aperture (25) can be scanned by adjusting the scanner (12) with the focus area (7) of the illuminating light (23) and the detection aperture (11) of the detector (8) can be scanned by adjusting the scanner (12) with the auxiliary light (22) from the auxiliary light source (20) can be scanned.

17. The device according to claim 11 or microscope assembly (3) according to claim 16, characterized in that

that the auxiliary light source (20) and the auxiliary detector (24) are the same photoelectric component (19), the photoelectric component (19) optionally being a light emitting diode (50), a super luminescent diode, a laser diode (43) or a photodiode (42) , or

that the auxiliary detection aperture (25) of the auxiliary detector (24) and the auxiliary emission aperture (21) of the auxiliary light source (20) are formed with an end cross section (39) of a light guide (40) arranged in the focal plane (6), the light guide (40) being optional is branched via a free beam beam splitter, a fiber beam splitter or a circulator (41) to the auxiliary light source (20) and the auxiliary detector (24).

18. microscope assembly (3) according to one of claims 12 to 15 for performing the method according to claim 9 with

the light source (4) providing the illuminating light (23),

the scanner (12) between the light source (4) and the detector (8) on the one hand and the focal plane (6) on the other hand, and

the auxiliary detector (24) to which the illuminating light (23) can be directed via the scanner (12), characterized in that the auxiliary detection aperture (25) of the auxiliary detector (24) is arranged in the focal plane (6) and the auxiliary emission aperture (21 ') of the separate auxiliary light source (20') providing the auxiliary light (22) is so concentric to the detection aperture (11) ) of the detector (8) is arranged that the detection auxiliary aperture (25) of the auxiliary detector (24) by adjusting the scanner (12) on the one hand with the focus area (7) of the illuminating light (23) and on the other hand with that from the emission auxiliary aperture (21 ') the auxiliary light (22') emerging from the auxiliary light source (20 ') can be scanned.

19. microscope assembly (3) according to one of claims 12 to 15 for performing the method according to claim 10 with

the detector (8) for the light from the focus area (7) which has the detection aperture (11) to be arranged confocally to the focus area (7),

characterized in that the auxiliary emission aperture (21) of the auxiliary light source (20) through which the auxiliary light (22) emerges is arranged in the focal plane (6) and the auxiliary detection aperture (25 ') of the separate auxiliary detector (24') is so concentric the emission aperture (65) of the light source (4) is arranged that on the one hand the detection auxiliary aperture (25 ') of the separate auxiliary detector (24') and on the other hand the detection aperture (11) of the detector (8) by adjusting the scanner (12) the auxiliary light (22) can be scanned by the auxiliary light source (20).

20. Laser scanning microscope with an objective and a scanning and descanning microscope assembly (3) according to one of claims 12 to 19.

21. Laser scanning microscope according to claim 20, characterized in that that the detection auxiliary aperture (25) of the auxiliary detector (24) and / or the

Emission auxiliary aperture (21) of the auxiliary light source (20) are arranged in an intermediate image plane (34) of the laser scanning microscope (1) or

that the detection auxiliary aperture (25) of the auxiliary detector (24) and / or the

Emission auxiliary aperture (21) of the auxiliary light source (20) are arranged in a branch (16) from a main beam path (17) of the laser scanning microscope (1), with optional

the branch (16) branches off and / or on a beam splitter (18), a deflecting mirror (34) or a rotating mirror (13) of the scanner (12) in the main beam path of the laser scanning microscope (1)

22. Scanning and descanning microscope assembly (3) according to one of claims 12 to 19 or laser scanning microscope according to one of claims 20 and 21, characterized in that the detection aperture (11) of the detector (8) through a pinhole arranged in front of the detector (8) (10) is limited.

23. Scanning and descanning microscope assembly (3) according to one of claims 12 to 19 and 22 or laser scanning microscope according to one of claims 20, 21 and 22, characterized in that the detector (8) is a point detector or an array detector.